This project uses genetic, biochemical and structural approaches to understand how autonomously replicating parvoviruses target and enter their host cells and establish infection. It will provide new information on the control of tissue tropis and on structural transitions in the virion that regulate successive steps in the infectious entry pathway of these ubiquitous viruses, particularly relating to an unexpected mode of genome exposure that could allow the capsid to fulfill novel intranuclear functions. The autonomously replicating parvoviruses are rugged and genetically simple single-stranded DNA viruses that are non-transforming because they are unable to activate resting cells to re-enter the cell cycle, and thus depend upon the host cell's regulation of cell cycle progression. In addition, many parvovirus species that infect rodents are inherently oncoselective, and preferentially infect transformed human cells, suggesting that they could be developed as therapeutic agents to target human tumors such as melanoma. The project pursues four approaches to understanding and exploiting such target specificity. 1. The targeting functions of the parvoviral shell will be identified by mapping the fortuitous tropism of parvovirus LuIII for human melanoma cells. This will involve making capsid chimeras with the non- melanotropic murine virus. Since LuIII is unlikely to be optimally melanotropic, we will use DNA shuffling to ask whether this property can be enhanced. 2. Differential qPCR analysis of sub-fractions of infected cells, expression of dominant-negative host genes and a novel in situ hybridization approach will be used to follow virions as they hijack specific intracellular trafficking pathways to gain access to the host nucleus. 3. Collaborative structural studies will be combined with genetic and biochemical approaches to investigate sequential conformational changes in the virion that facilitate ordered progress through its lifecycle. We have shown that genome uncoating is not a simple reversal of the packaging process, and will use these combined approaches to determine how the structurally symmetrical parvoviral capsid packages, retains and then releases its genome in the appropriate cellular compartments. 4. Optimized capsids will be used to package vectors designed to deliver heterologous genes into human and mouse melanoma cells. We will test the efficacy of a parvoviral vector packaged in an appropriately optimized capsid for targeting a transplantable murine melanoma in vivo. The vector expresses the B7-1 co-stimulatory molecule, and will potentially activate cytotoxic killer T-cells specific for melanoma tumor antigens, resulting in tumor eradication.